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Originally published In Press as doi:10.1074/jbc.M005464200 on August 1, 2000

J. Biol. Chem., Vol. 275, Issue 43, 33869-33875, October 27, 2000
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Evolution of the adhE Gene Product of Escherichia coli from a Functional Reductase to a Dehydrogenase
GENETIC AND BIOCHEMICAL STUDIES OF THE MUTANT PROTEINS*

Jorge Membrillo-HernándezDagger §, Pedro Echave||, Elisa Cabiscol, Jordi Tamarit**, Joaquim RosDagger Dagger , and Edmund C. C. LinDagger

From the Dagger  Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 and  Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Spain

The multifunctional AdhE protein of Escherichia coli (encoded by the adhE gene) physiologically catalyzes the sequential reduction of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. The NH2-terminal region of the AdhE protein is highly homologous to aldehyde:NAD+ oxidoreductases, whereas the COOH-terminal region is homologous to a family of Fe2+-dependent ethanol:NAD+ oxidoreductases. This fusion protein also functions as a pyruvate formate lyase deactivase. E. coli cannot grow aerobically on ethanol as the sole carbon and energy source because of inadequate rate of adhE transcription and the vulnerability of the AdhE protein to metal-catalyzed oxidation. In this study, we characterized 16 independent two-step mutants with acquired and improved aerobic growth ability on ethanol. The AdhE proteins in these mutants catalyzed the sequential oxidation of ethanol to acetaldehyde and to acetyl-CoA. All first stage mutants grew on ethanol with a doubling time of about 240 min. Sequence analysis of a randomly chosen mutant revealed an Ala-267 right-arrow Thr substitution in the acetaldehyde:NAD+ oxidoreductase domain of AdhE. All second stage mutants grew on ethanol with a doubling time of about 90 min, and all of them produced an AdhEA267T/E568K. Purified AdhEA267T and AdhEA267T/E568K showed highly elevated acetaldehyde dehydrogenase activities. It therefore appears that when AdhE catalyzes the two sequential reactions in the counter-physiological direction, acetaldehyde dehydrogenation is the rate-limiting step. Both mutant proteins were more thermosensitive than the wild-type protein, but AdhEA267T/E568K was more thermal stable than AdhEA267T. Since both mutant enzymes exhibited similar kinetic properties, the second mutation probably conferred an increased growth rate on ethanol by stabilizing AdhEA267T.

* This work was supported by United States Public Health Service Grant GM40993 from the NIGMS of the National Institutes of Health and Dirección General de Enseñanza Superior e Investigación Científica Project PB97-1456.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of The Bernard D. Davis Fellowship. Present address: Dept. de Biología Molecular, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, P. O. Box 70-228, 04510 Mexico City, México.

|| Recipient of a Ph.D. fellowship from the Ministerio de Educación y Cultura (Spain).

** Recipient of a postdoctoral fellowship from the Generalitat de Catalunya (Spain).

Dagger Dagger To whom correspondence should be addressed. Tel.: 34 973 702 407; Fax: 34 973 702 426.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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